The zebrafish has emerged as a premier model organism for the study of human development and disease. Its greatest asset is that it is a genetic system in which mutant phenotypes can be effectively dissected at the organismal, cellular and subcellular levels. Many forward genetic screens have identified genes and subsequently delineated their roles in embryonic cell fate specification, patterning, morphogenesis and organogenesis. However with the sequencing of the zebrafish genome we now know that many genes have never been identified by forward genetics, so their role in development remains an open question. Furthermore, recent explosion in the identification of human disease-causing genes has the potential to re-purpose the zebrafish as a leading model for human genetic disease, provided the zebrafish homologs of these genes can be mutated within the next few years. A number of reverse genetics approaches are currently being pursued in the zebrafish, with the potential to generate knock-outs in many of the genes in the genome. However, these approaches are largely untargeted and therefore do not address the immediate and pressing needs of the scientific community. We have created a large library of 15,456 cryopreserved, ENU mutagenized fish and estimate that this library contains at least one nonsense allele in any average-sized zebrafish gene with 97% chance. We have developed methodologies to screen this library efficiently for mutations that are present in a single heterozygous fish, and using these methodologies we have identified one or more nonsense mutations in over 80 genes considered to be of high-priority by zebrafish researchers. We have distributed these mutants widely within the community, resulting in 18 publications in the past four years. We have now transitioned to innovative and high-throughput Illumina sequencing methodologies for mutation detection with which we propose to identify one or more loss-of-function mutation in over 300 zebrafish genes in three years, and to deliver them to the community via ZIRC.